Implantation of the embryo into the uterus and the resultant formation of the placenta are unique and critical features of mammalian development. Implantation is mediated by the invasive behavior of the trophoblast cells, which differentiate from the trophectoderm cells of the blastocyst. Trophoblast invasive behavior is developmentally regulated; the precursor trophectoderm cells are quiescent epithelial cells which acquire motile invasive behavior with differentiation. The goals of this proposal are to examine and define the changes in cell behavior that accompany trophoblast differentiation, and, following the paradigms recently defined for the control of cell motility in cultured cells, to test the role of the Rho family of small GTP-binding proteins in the developmental regulation of trophoblast cell behavior. Time-lapse video analysis of embryos cultured in normal medium and in the presence of inhibitors will be used to determine when motile behavior is normally initiated, and at what point in development transcription and translation are required for its onset. The results will be compared to analyses of trophoblast adhesivity and molecular differentiation, to determine the relationship between these components of invasive behavior. These results will better define the process of trophoblast differentiation, and provide the foundation for interpreting and extending the transgenic experiments. Members of the Rho family of GTP-binding proteins have been shown in cultured cells to regulate formation of filopodial protrusions (Cdc42Hs), Iamellipodia (Rac) and stress fibers and focal contacts (Rho). To determine the role of these three proteins in the regulation of trophoblast cell motility, gene transfer through recombinant adenovirus infection will be used to overexpress dominant negative or constitutively active forms of each protein in preimplantation stage embryos. The dominant negative overexpression experiments will demonstrate whether each of the Rho family members is involved in the control of trophoblast motility, while the constitutively active overexpression experiments will address whether these proteins serve as control points for temporal regulation of trophoblast motility. The results of these experiments will clarify the mechanisms regulating trophoblast behavior and differentiation, and will provide a model both for later, less accessible developmental events, and for invasive transformation in other systems.